The galectins are a family of proteins defined by shared sequence elements and by affinity for β-galactosides (Barondes et al., 1994). There are now ten known mammalian galectins (FIG. 1), but biochemical analysis of tissues as well as the accumulation of partial DNA sequences from expressed sequence tags (ESTs) suggest that there are many more (Cooper and Barondes, 1999). Galectins occur at high concentration (usually 0.–1% of total soluble cell protein) in a limited range of cell types, different for each galectin.
All galectins bind lactose and other β-galactosides, but they differ in their affinity for more complex saccharides (Leffler and Barondes, 1986, Barondes et al., 1994). This suggests that galectins may play a role in decoding the information in complex carbohydrates at the cell surface and in the extracellular matrix. A review of the data up to 1999 is given by Leffler (2001). By cross-linking cell-surface and extracellular glycoproteins (e.g. laminin, integrins, and IgE receptors), extracellular galectins are known to modulate cell adhesion and induce intracellular signals. By the adhesion modulation, galectins may play roles in maintenance of tissue integrity and in cancer metastasis. By the signaling activity, galectins may induce a variety of responses including apoptosis in T-lymphocytes, oxidative burst in neutrophil leukocytes, and through these activities be important in inflammation and immune regulation. In addition, galectins may have intracellular functions; there is evidence for binding to intracellular non-carbohydrate ligands, and roles in RNA splicing and modulation of apoptosis have been suggested.
The best studied are galectin-3 and galectin-1. The present invention relates mainly to galectin-3, but its principles may be applicable also to other galectins.
Potential Therapeutic Use of Galectin-3 Inhibitors. Galectin-3 has been implicated in diverse phenomena and, hence inhibitors may have multiple uses. It is easy to perceive this as a lack of specificity or lack of scientific focus. Therefore, the analogy with aspirin and the cyclooxygenases (COX-I and II) is useful. The COXs produce the precursor of a wide variety of prostaglandins and, hence, are involved in a diverse array of biological mechanisms. Their inhibitors, aspirin and other NSAIDs (non-steroid anti-inflammatory drugs), also have broad and diverse effects. Despite this, these inhibitors are very useful medically, and they have several different specific utilities.
So if galectins, like COXs, are part of some basic biological regulatory mechanism (as yet unknown), they are likely to be ‘used by nature’ for different purpose in different contexts. Galectin inhibitors, like NSAIDs, are not expected to wipe out the whole system, but to tilt the balance a bit.
Inhibition of Inflammation.
There is now ample evidence that galectin-3 is proinflammatory (reviewed by Leffler, 2001). Its expression is induced in macrophages and other cells during inflammation (Perillo et al., 1998). It has various proinflammatory effects on other cells in the inflammatory site (Sano et al., 2000; Karlsson et al., 1998). Galectin-3 gene null-mutant (knock-out) mice have decreased inflammatory responses (Hsu et al., 2000) and knock-out mice of Mac-2BP, a galectin-3 ligand, have increased inflammatory responses (Trahey et al., 1999). Inflammation is a protective response of the body to invading organisms and tissue injury. However, if unbalanced it also frequently is destructive and occur as part of the pathology in many diseases. Because of this there is great medical interest in pharmacological modulation of inflammation. A galectin-3 inhibitor is expected to provide an important addition to the arsenal available for this.
Treatment of Septic Shock.
The idea of a possible role of galectin-3 in septic shock comes from our own studies (Almquist et al., 2001). Briefly the argument goes as follows. It is known that septic shock involves dissemination of bacterial lipopolysaccharide into the blood stream, and that the pathological effects of this are mediated via neutrophil leukocytes (Karima et al., 1999). LPS does not activate the tissue damaging response of the neutrophil. Instead it primes the neutrophil, so that it is converted from unresponsive to responsive to other, presumably endogenous, activators. In septic shock this priming happens prematurely in the blood stream. Endogenous activators could then induce the tissue damaging response in the wrong place and time. Several candidates have been proposed as these endogenous activators, including TNF-alfa. Inhibitors of these have been used in treatment schemes without much success (Karima et al., 1999). Since our own studies indicate that galectin-3 is a good candidate as an endogenous activator of primed neutrophils (Almquist et al., 2001), galectin-3 inhibitors may be very useful in septic shock.
Treatment of Cancer.
There is a whole other body of evidence suggesting that induced expression of galectin-3 (and perhaps other galectins) promote tumour growth and/or metastasis (reviewed by Leffler, 2001). The evidence is on one hand correlatory—more galectin in more malignant tumours. The direct evidence comes from animal models, mainly by Raz et al, but also others. In paired human tumour cell lines (with decreased or increased expression of galectin-3), the one with more galectin-3 gives more tumours and metastasis in nude mice (Bresalier et al., 1998). A polysaccharide, which inhibits galectin-3 can inhibit tumours in vivo (Pienta et al., 1995). Although there may be different explanations for the effects of galectin-3, inhibition of its activities is expected to be beneficial in cancer.
Galectin-1 and galectin-9 have been shown to induce apoptosis in activated T-cells. Also, galectin-1 is frequently over-expressed in low differentiated cancer cells, and galectin-9 (or its relatives galectin-4 and galectin-8) is expressed in certain cancer types. Hence, these galectins might help the tumour to defend itself against the immune response raised by the host (Perillo et al., 1998; Leffler, 2001). Inhibitors of the galectin would be expected to block such an effect and thereby be useful in cancer treatment.
Known Inhibitors
Natural Ligands.
Solid phase binding assays and inhibition assays have identified a number of saccharides and glycoconjugates with the ability to bind galectins (reviewed by Leffler, 2001). All galectins bind lactose with Kd of 0,5–1 mM. The affinity of D-galactose is 50–100 times lower. N-Acetyllactosamine and related disaccharides bind about as well as lactose but for certain galectins up to 10 times better. The best small saccharide ligands for galectin-3 were those carrying blood group A-determinants attached to lactose or lacNAc-residues and were found to bind up to about 50 times better than lactose. Galectin-1 shows no preference for these saccharides.
Larger saccharides of the polylactosamine type have been proposed as preferred ligands for galectins. In solution using polylactosamine carrying glycopeptides, there was evidence for this for galectin-3 but not galectin-1 (Leffler and Barondes, 1986). A modified plant pectin polysaccharide has been reported to bind galectin-3 (Pienta et al., 1995).
The above described natural saccharides that have been identified as galectin-3 ligands are not suitable for use as active components in pharmaceutical compositions, because they are susceptible to acidic hydrolysis in the stomach and to enzymatic degradation. In addition, natural saccharides are hydrophilic in nature and are not readily absorbed from the gastrointestinal tract following oral administration.
Synthetic Inhibitors.
Thiodigalactoside is known to be a synthetic inhibitor approximately as efficient as N-acetyllactosamine (Leffler and Barondes, 1986). Saccharides coupled to amino acids with anti-cancer activity were first identified as natural compounds in serum, but subsequently synthetic analogues have been made (Glinsky et al., 1996). Among them, those with lactose or Gal coupled to the amino acid inhibits galectins but only with about the same potency as the corresponding underivatized sugar. A divalent form of a lactosyl-amino acid had higher potency in a solid phase assay (Naidenko et al., 2000). Starburst dendrimers (André et al, 1999) and glycopolymers (Pohl et al, 1999), made polyvalent in lactose-residues, have been described as galectin-3 inhibitors with marginally improved potency as compared to lactose. The aforementioned synthetic compounds that have been identified as galectin-3 ligands are not suitable for use as active components in pharmaceutical compositions, because they are hydrophilic in nature and are not readily absorbed from the gastrointestinal tract following oral administration. Dendrimers and glycopolymers are too large to be absorbed and large enough to produce immune responses in patients. Furthermore, dendrimers and glycopolymers are susceptible to acidic hydrolysis in the stomach and to enzymatic hydrolysis.
Thus, there is a considerable need within the art of inhibitors against galectin, in particularly to galectin 3.